WANG Qian-nan,HUANG Pan-pan,ZHOU Shan-yue

The Key Lab of Integrated Crop Pests Management of Shandong Province/College of Plant Health and Medicine,Qingdao Agricultural University,Qingdao 266109,P.R.China

Abstract FgGCN5,a GCN5 homolog in Fusarium graminearum,plays a critical role in hyphal vegetative growth,asexual and sexual reproduction,deoxynivalenol (DON) biosynthesis and plant infection.For nuclear localized GCN5,four conserved sequence motifs (I-IV) are presented in the catalytic domain and a bromodomain in the carboxy-terminus.As a lysine acetyltransferase,conserved negatively charged residues are present to neutralize the protons from lysine substrates.However,the role of conserved motifs/domains and residues in FgGCN5 are unclear.Here,we generated deletion mutant strains for each the conserved motifs/domains and a glutamate residue 130 (E130) replacement mutant.Deletion of each conserved motif in the catalytic domain and replacement of E130 site resulted in manifold defects in hyphae growth,asexual and sexual development,DON biosynthesis,and plant infection.Phenotypic defects in the mutant strains were similar to deletion mutants.The deletion of the bromodomain led a significant reduction in DON production and virulence,with no effects on hyphae growth,asexual or sexual reproduction.FgGCN5 was further found to localize to the nucleus in conidia and hyphae cells.In conclusion,FgGCN5 encodes a nuclear localized acetyltransferase.The conserved motifs in the catalytic domain and E130 are essential for correct functions of the gene.The conserved bromodomain is important for DON production and pathogen virulence.This was the first report to identify the functions of conserved motifs/domains in FgGCN5,which will contribute to our understanding of the mechanism(s) by which FgGCN5 regulates F.graminearum.

Keywords:FgGCN5,catalytic domain,bromodomain,DON,virulence

1.lntroduction

Lysine acetylation is a conserved post-translational modification (PTM) occurring ubiquitously both in eukaryotes and prokaryotes.The modification is catalyzed by lysine acetyltransferases (KATs) and reversed by lysine deactylases (KDACs) (Kuo and Allis 1998; Henriksenet al.2012).Dependent on their localization in cells,KATs are divided into two major classes,nuclear A-type and cytoplasmic B-type (Dawson and Kouzarides 2012).According to their sequence conservation and structure,KATs are grouped into different families,including GNAT,MYST,and Rtt109 (Elias-Villaloboset al.2019).GCN5 belongs to the GNAT family and has been well studied.Sequence analysis revealed that within GCN5,four conserved sequence motifs (I-IV) are presented in the catalytic domain and amino-terminus,with conserved bromodomains in the carboxy-terminus that are absent in B-type KATs (Brownellet al.1996).Lysine acetylation plays a critical regulatory role in almost all cellular process.As a representative,homologs of GCN5 play important roles in fungal growth,development,secondary metabolism,and virulence (Cánovaset al.2014; Gonzalez-Prietoet al.2014;Changet al.2015; Lanet al.2016; Rosleret al.2016).

Fusarium graminearumis a destructive fungal pathogen that causes Fusarium head blight (FHB) on small grain cereals (Honget al.2010; Adnanet al.2019).Infection of the pathogen leads to substantial yield loss.More seriously,during plant infection,the pathogen produces mycotoxins that are harmful to humans and livestock (Bennettet al.2003; McMullenet al.2012).To provide global insight into the roles of lysine acetylation inF.graminearum,systematic acetylome analysis was performed,which was suggestive of lysine acetylation during DON production and virulence (Zhouet al.2016).For further investigation of the roles of lysine acetylation,KATs were functional characterized (Konget al.2018).FgGCN5,theGCN5homolog ofF.graminearum,was confirmed to play a critical role in hyphal vegetative growth,conidiation,DON biosynthesis,sexual reproduction and plant infection(Chenet al.2018; Konget al.2018; Zhou and Wu 2019).However,the correlations between the conserved motifs/domains and the functionality ofFgGCN5have remained unclear.

In this study,to provide insight into the functions of the conserved motifs/domains in FgGCN5,the conserved motifs/domains deletion mutants were generated.E130K site mutations and deletion mutants in the conserved motifs of the catalytic domain of FgGCN5 showed defects in mycelium growth,conidiation,DON production,sexual reproduction and plant infection,with symptoms similar to those observed for gene deletion mutants.The deletion of the conserved bromodomain in the carboxy-terminus of FgGCN5 resulted in a significant reduction in DON production and virulence,however,the deletion had an undetectable impact on the mycelium growth,conidiation and sexual reproduction,compared to the wild type.Moreover,qRT-PCR revealed that the transcription ofTrigenes with key roles in DON biosynthesis were enhanced in the bromodomain deletion mutant.To our knowledge,this is the first report to link functions and conserved motifs/domains in FgGCN5,which lays foundation for an enhanced understanding ofFgGCN5inF.graminearum.

2.Materials and methods

2.1.Strains and culture conditions

Wild type PH-1,gene deletion mutants,conserved motifs/domain deletion mutants,site-directed mutants and the complementary strains ofF.graminearumwere cultured on PDA medium at 25°C.Hyphal growth rates of the different strains were examined as previously described (Zhouet al.2010).Protoplast preparation and the transformation ofF.graminearumwere performed following previously reported methods (Houet al.2002; Liet al.2011,2018).

2.2.Conserved motifs/domain analysis in FgGCN5

The conserved motifs of the catalytic domain in the N-terminus of FgGCN5 were analyzed by protein alignment(Linet al.1999).The conserved bromodomain in the C-terminal of FgGCN5 was analyzed with SMART (http://www.expasy.org/tools).

2.3.Generation of the conserved motifs/domains deletion mutants,site-directed mutants,gfp-FgGCN5 fusions and ΔFgGCN5-C complementary strains

To generate the conserved motifs/domains deletion mutants,upstream and downstream DNA sequences inFgGCN5were amplified.Specific primers were designed:cFgGCN5F/FDr and FDf/cFgGCN5R for full-length catalytic domain deletion mutantsFgGCN5ΔN; cFgGCN5F/Ir and If/cFgGCN5R for the deletion of conserved motif I mutantsFgGCN5ΔI; cFgGCN5F/IIr and IIf/cFgGCN5R for the deletion of conserved motif II mutantsFgGCN5ΔII; cFgGCN5F/IIIr and IIIf/cFgGCN5R for deletion of conserved motif III mutantsFgGCN5ΔIII; cFgGCN5F/IVr and IVf/cFgGCN5R for the deletion of conserved motif IV mutantsFgGCN5ΔIV;cFgGCN5F/DB5r and DB3f/cFgGCN5R for the deletion of the conserved bromodomain deletion mutantsFgGCN5ΔC.For the FgGCN5E130Kmutant,the upstream and downstream sequences ofFgGCN5were amplified with specific primers cFgGCN5F/Er and Ef/cFgGCN5R.To generate the complementary strainΔFgGCN5-C,theFgGCN5fragment was amplified with primers cFgGCN5F and cFgGCN5R.Finally,DNA fragments for the corresponding mutants and linear pFL2 digested withXhoI were co-transformed into competent cells of yeast strain XK1-25 following previously described methods (Jianget al.2016).Plasmids were confirmed by sequencing and used to transformΔFgGCN5protoplasts.The gene deletion mutantΔFgGCN5was constructed by us previously (Zhou and Wu 2019).Transformants with geneticin resistance were further confirmed by qRT-PCR with JDf/JDr primers.

Forgfp-FgGCN5mutants,fragments ofFgGCN5were amplified withNGf (containing theEcoRI sites at the 5′terminus) and NGr (containing theBamHIsites at the 5′terminus).Following digestion with the corresponding restriction endonuclease,the resulting PCR products were inserted into pKNGFP vectors at the 3′-terminal end ofgfp,so thegfp-FgGCN5fusions could be expressed under the control of the PtrpC promoter.Plasmids were confirmed by sequencing and transformed intoΔFgGCN5protoplasts.Transformants were confirmed following the methods to generate the motifs deletion mutants.Primers used to construct the mutants are listed in Table 1.

2.4.Conidiation assays

For conidiation assays,six mycelial plugs (9 mm in diameter)of each strain were taken from the periphery of a 4-day-old colonies and cultured in 100-mL flasks containing 50 mL of 3% mung bean broth (Chenet al.2009; Qinet al.2020).After 5 days of incubation at 25°C with shaking at 180 r min-1,conidia were harvested by filtration (Qinet al.2020).After centrifugation at 5 000 r min-1at room temperature for 10 min,conidia were re-suspended in 1 mL of sterile distilled water and counted on a hemocytometer.The morphology of conidia in each strain was observed through imaging.Three independent repetitions were performed and at least 200 conidia were examined for each repetition.

2.5.Plant infection assays

For infection assays,the conidia of each strain were collected from 5-day-old mung bean liquid medium and re-suspended to 2.0×105conidia mL-1in sterile water.For inoculation,the flowering wheat heads of cultivar Qingmai 6 were used.The third spikelet from the base of each head was drop-inoculated into 10 μL of conidial suspensions as previously described (Galeet al.2002,2007; Lysoeet al.2011; Liet al.2015; Jianget al.2016).For mutant strains defective in conidia production,mycelial suspensions were used as the inoculum (Konget al.2018).Each treatment was performed on three independent occasions and 15 wheat heads were inoculated into single treatment for each strain.The disease index of each strain was estimated after 14 days of infection (Dinget al.2009; Wang C Fet al.2011;Howlettet al.2012).

Infection assays were performed with corn silk as previously described (Seonget al.2005).Fresh corn silks were collected and sliced into～5 cm fragments.Seven to ten corn silk fragments were aligned to a bundle and added evenly onto Whatman no.1 filter paper soaked with sterile water.The lower terminal of the corn silk bundle was covered with mycelium plugs taken from the periphery of 3-day-old colonies of each strain.Corn silks were incubated at 25°C under high humidity.After incubation for 5 days,infections were scored based on the discoloration of the corn silk.

2.6.DON production assays

As some mutant strains are defective in conidia production,DON production assays were performed with uniform mycelial suspensions of each strain in TBI medium (Gardineret al.2009).The mycelial suspensions of each strain were incubated as described by Jianget al.(2016).After 7 days of incubation,cultures of each strain were filtered with sterile miracloths.Collections were filtered through a C18/neutral alumina (1:3,w/v) column.Filtrates were dried in a vacuum and the resulting residues were re-dissolved in TMS (TMSI:TMCS=100:1).After the resuspensions were fully mixed with a vortex,isooctane was added for DON extraction.Supernatants were collected and DON was detected by the gas chromatography-mass spectrometer(GC-MS).DON production of each strain was finally determined according to three experimental repeats.

Table 1 Primers for construction and identification of mutants in the study

2.7.RNA extraction and qRT-PCR analysis

Total RNA was extracted and TRI genes were detected by qRT-PCR according to the methods described by Jiang(2016).Briefly,mycelium of each strain were incubated in TBI medium at 25°C with shaking at 180 r min-1in the dark.After incubation for 3 days,the mycelial of each strain were harvested by centrifugation.RNA extractions for each strain were performed with TRIzol (Invitrogen,USA).Extracted RNAs were used for first strand cDNA synthesis by reverse transcription with TaKaRa PrimeScriptTMRT Reagent Kits(TaKaRa Bio Inc.,Dalian,China).The expressions ofTRI5,TRI6andTRI10,all of which are essential for trichothecene biosynthesis were examined by qPCR,whilst the betatubulin geneTub2of the fungus was used as an internal control.Relative gene expression was calculated using the 2-ΔΔCtmethod (Livak and Schmittgen 2001).The mean and standard deviation were determined from three independent replicates.

2.8.Sexual reproduction assays

Sexual reproduction assays were performed as previously described (Wang Yet al.2011; Zhenget al.2013).Briefly,mycelial plugs (5 mm in diameter) were taken from the margins of 3-day-old colonies of each strain which was inoculated onto the carrot medium.Seven days later,aerial hyphae of each strain were pressed with sterile 0.1% Tween 20.One week later,perithecia,cirrhi,asci,and ascospore discharge of each strain were observed (Cavinderet al.2012; Luoet al.2014; Caoet al.2017).

2.9.Subcellular localization of FgGCN5

For subcellular localization assays in live cells,gfp-FgGCN5fusions were constructed.The conidia and hyphae ofgfp-FgGCN5was stained with Hoechst 33342 (Gomeset al.2018).Conidia and hyphae of the strains were observed by fluorescence microscopy.

2.10.Statistical analysis

All data for hyphal growth,conidiation,DON production,and disease indexes of the strains were obtained from three independent replicates.Statistical analyses were performed using one-way ANOVA followed by Duncan's multiple range tests (P≤0.05).

3.Results

3.1.Conserved sequence motifs/domains in FgGCN5

It has been documented thatFgGCN5plays a key role in vegetative growth,asexual and sexual reproduction,plant infection,and DON production inF.graminearum(Chenet al.2018; Konget al.2018; Zhou and Wu 2019).In this study,to characterize the role of the conserved motifs/domains in FgGCN5,we derived the sequences of GCN5 of different organisms according to their accession numbers from the NCBI database.Multiple sequence alignments were performed for the different GCN5 proteins.Conserved motifs I to IV in the catalytic domain were confirmed (Fig.1).The conserved bromodomain in the carboxy-terminal region was predicted from the 286th to 386th amino acid by SMART.

3.2.Roles of the different motifs/domains in vegetative growth and conidiation

To characterize the roles of conserved motifs I-IV in the amino-terminal catalytic domain,the bromodomain in the carboxy-terminus,motifs I-IV and the bromodomain were deleted respectively.The glutamate residue 130 (E130)is conserved in GCN5 homologs as a proton acceptor,possessing the potential to regulate the catalytic activity of FgGCN5.Thus,the E130 in FgGCN5 was replaced by lysine (K) in this study.FgGCN5ΔI,FgGCN5ΔII,FgGCN5ΔIII,FgGCN5ΔIV,FgGCN5ΔNand theFgGCN5E130Kmutant strains developed colonies of similar morphology on PDA medium(Fig.2-A).Moreover,in vegetative growth assays,mutant strains showed a significant reduction in mycelium growth rates compared to the wild type and the complementary strainΔFgGCN5-C(Fig.2-B).Furthermore,the morphology and mycelium growth rates of the mutant strains were similar to those of the gene deletion mutantΔFgGCN5.To confirm the defects of the mutant strains in terms of colony morphology and hyphal growth resulting from the conserved motifs,gene expression was assessed by RT-PCR (Fig.3).The bromodomain deletion mutantFgGCN5ΔCdisplayed similar colony morphologies (Fig.2-A) and mycelium growth rates to the wild type and the complementary strainΔFgGCN5-C(Fig.2-B).The results revealed that conserved motifs I-IV in the amino-terminal catalytic domain,and conserved E130 in the FgGCN5 plays important roles in vegetative growth inF.graminearum,whilst the bromodomain was dispensable for the vegetative growth of the fungus.

Fig.1 Conserved sequence motifs/domains in the schematic drawing of FgGCN5.A,the schematic drawing of the FgGCN5.B,alignment was performed with the GCN5 proteins of different organisms derived from the protein NCBI database.The conserved sequence motifs I-IV in the catalytic domain of FgGCN5 are underlined and the conserved E130 is marked with ＊.

Fig.2 Colonies and the hyphal growth rate of the mutant strains.A,the colonies of the strains formed on the PDA medium.B,the growth rate of the hyphae of each strain assayed after incubation for 3 days at 25°C.Columns with different letters represent significant difference (P≤0.05).Bars mean standard deviation (n=3).ΔFgGCN5,the FgGCN5 deletion mutant; ΔFgGCN5-C,the complementary strain of the gene deletion mutant; FgGCN5ΔC,the bromodomain deletion mutant; FgGCN5ΔN,the catalytic domain deletion mutant; FgGCN5E130K,E130K site mutant; FgGCN5ΔI-FgGCN5ΔIV,the motifs I-IV deletion mutants.

For conidiation assays,the conidia of the mutant strains were harvested from mung bean broth cultures.As shown in Fig.4,the deletion of the conserved domains I-IV in the amino-terminal catalytic domain,and replacement of E130 in FgGCN5 resulted in a failure of conidiation in the mutants.However,deletion of the conserved bromodomain did not affect conidiation in the mutant.These results reveal that the conserved domains I-IV in the amino-terminal catalytic domain,and conserved E130 in FgGCN5 are critical for conidiation.

3.3.Roles in plant infection

After inoculation for 14 days,scab symptoms on the wheat heads were detected.As shown in Fig.5-A,FgGCN5ΔI,FgGCN5ΔII,FgGCN5ΔIII,FgGCN5ΔIV,FgGCN5ΔNandFgGCN5E130Kmutant strains were non-pathogenic on the wheat as determined through gene deletion mutantΔFgGCN5.Typical scab symptom were observed only on the wheat heads inoculated with the wild type,FgGCN5ΔCandΔFgGCN5-Cstrains.The disease indexes (DI) were calculated for each strain.The results showed no significant differences in pathogenicity betweenΔFgGCN5-C(DI=6.7±0.6) and the wild type strain (DI=7.05±0.85).However,a significant difference in the pathogenicity betweenFgGCN5ΔC(DI=4.1±0.7) and the wild type strain was observed.In infection assays with corn silk,the results were consistent with those of infection assays on the wheat heads.Diseased corn silk caused byFgGCN5ΔCwas shorter than that caused by wild type strains,andFgGCN5ΔI,FgGCN5ΔII,FgGCN5ΔIII,FgGCN5ΔIV,FgGCN5ΔNandFgGCN5E130Kmutant strains were non-pathogenic on the corn silk,as observed forΔFgGCN5(Fig.5-B).These results suggest that the conserved bromodomain is critical to pathogen virulence,whilst conserved motifs I-IV in the catalytic domain and E130 are essential to plant infection.

Fig.3 Transcription of the truncated genes detected by RT-PCR with total RNAs extracted from the hyphae of each mutant strain.Lines 1-10 represent fungal strains PH-1,ΔFgGCN5,ΔFgGCN5-C,FgGCN5ΔC,FgGCN5ΔN,FgGCN5E130K,FgGCN5ΔI-FgGCN5ΔIV,respectively.

3.4.Roles in DON production

Fig.4 Conidiation of the strains cultured in mung bean liquid medium.The mycelium plugs of each strain were incubated in medium for 5 days,and then the conidia were harvested and the conidia number were counted by a haemocytometer.ΔFgGCN5,the FgGCN5 deletion mutant; ΔFgGCN5-C,the complementary strain of the gene deletion mutant; FgGCN5ΔC,the bromodomain deletion mutant; FgGCN5ΔN,the catalytic domain deletion mutant; FgGCN5E130K,E130K site mutant;FgGCN5ΔI-FgGCN5ΔIV,the motifs I-IV deletion mutants.Columns with different letters represent significant difference(P≤0.05).Bars mean standard deviation (n=3).

Fig.5 Virulence of the strains on wheat heads and corn silks.A,scab symptoms on wheat heads observed 14 days post inoculation with each strain.Fifteen wheat heads were inoculated in one repetition.B,diseased symptoms on corn silks 5 days post inoculation with each strain.Six bundles of corn silks were inoculated in one repetition.Each treatment was performed for three independent repetitions for inoculation both on heads and corn silks.ΔFgGCN5,the FgGCN5 deletion mutant; ΔFgGCN5-C,the complementary strain of the gene deletion mutant; FgGCN5ΔC,the bromodomain deletion mutant; FgGCN5ΔN,the catalytic domain deletion mutant;FgGCN5E130K,E130K site mutant; FgGCN5ΔI-FgGCN5ΔIV,the motifs I-IV deletion mutants.

Since DON production was inhibited inΔFgGCN5(Zhou and Wu 2019),DON production was assayed in motifs/domains mutants and E130 site mutants.DON production was blocked inFgGCN5ΔI,FgGCN5ΔII,FgGCN5ΔIII,FgGCN5ΔIV,FgGCN5ΔNandFgGCN5E130Kmutant strains,which recovered to wild type levels ((286.60±65.44) mg L-1) in theΔFgGCN5-Cstrain ((322.44±67.89) mg L-1).However,in theFgGCN5ΔCstrain,DON production ((116.09±7.51) mg L-1)was reduced compared to the wild type strains (Fig.6-A).These results indicate that the four conserved motifs in the catalytic domain and E130 are essential for DON production,and that the bromodomain plays an important role in DON biosynthesis.

The transcription ofTri5,Tri6,andTri10were assayed by qRT-PCR in the mutant strains.The results revealed that inFgGCN5ΔI,FgGCN5ΔII,FgGCN5ΔIII,FgGCN5ΔIV,FgGCN5ΔNandFgGCN5E130Kmutant strains,the expression ofTri5andTri6were undetectable,and thatTri10was expressed at a very low level.The expression of the three genes were comparable toΔFgGCN5.In the complementary strainΔFgGCN5-C,the expression of the three genes recovered to nearly wild type levels (Fig.6-B).InFgGCN5ΔC,the expression of the three genes were enhanced compared to the wild type,but the difference is not significant (Fig.6-B).

3.5.Roles in sexual development

After 8 days of incubation on carrot medium,FgGCN5ΔCandΔFgGCN5-Cproduced abundant perithecia and scospore cirrhi as observed for the PH-1 strain.No perithecia were observed fromFgGCN5ΔI,FgGCN5ΔII,FgGCN5ΔIII,FgGCN5ΔIV,FgGCN5ΔNand theFgGCN5E130Kmutant strains,which were similar to the defective phenotype of the gene deletion mutantΔFgGCN5(Fig.7-A).Moreover,abundant ascospores were discharged fromFgGCN5ΔCandΔFgGCN5-Cas observed for wild-type perithecia (Fig.7-B),in agreement with the observations of scospore cirrhi from perithecia.Furthermore,the asci and ascospores in perithecia were examined.As shown in Fig.7-C,most of the asci and ascospres produced byFgGCN5ΔCandΔFgGCN5-Cdeveloped into regular shapes that were comparable to the wild type.However,less than 8 ascospores in ascus were observed forFgGCN5ΔCandΔFgGCN5-Cstrains.These results indicate that sexual production recovered inΔFgGCN5-Cand thatthe bromodomain had no effect on the sexual production.Conserved domains I-IV in the catalytic zone and E130 residue were found to be essential for sexual production in the fungus.

3.6.Cellular localization

To confirm the subcellular localization of FgGCN5,gfp-FgGCN5 was generated.The GFP-FgGCN5 fusion localized to the nuclei of the conidia,evidenced by its colocalization with Hoechst 33342 (Fig.8-A).Likewise,the nuclear localization of the GFP-FgGCN5 fusion was observed in hyphal cells (Fig.8-B).These results suggest that FgGCN5 localizes to the nucleus in cells ofF.graminearum.

4.Discussion

4.1.The conserved motifs l-lV and E130 in catalytic domain are essential for the roles of FgGCN5

FgGCN5,aGCN5homolog inF.graminearum,plays critical roles in hyphae vegetative growth,DON biosynthesis,asexual and sexual reproduction,and plant infection.To gain further insight into the roles of the conserved motifs/domains in FgGCN5,we constructed mutant strains of each.

Fig.6 DON production of the strains and the relative transcription level of the Tri genes.A,DON production of the strains in TBI medium detected with gas chromatography-mass spectrometer (GC-MS).B,the transcription level of the Tri5,Tri6,and Tri10 examined by qRT-PCR.Columns with different letters represent significant difference (P≤0.05).ΔFgGCN5,the FgGCN5 deletion mutant; ΔFgGCN5-C,the complementary strain of the gene deletion mutant; FgGCN5ΔC,the bromodomain deletion mutant;FgGCN5ΔN,the catalytic domain deletion mutant; FgGCN5E130K,E130K site mutant; FgGCN5ΔI-FgGCN5ΔIV,the motifs I-IV deletion mutants.Bars mean standard deviation (n=3).

Fig.7 Sexual reproduction of the strains on carrot agar.A,perithecia produced by the conserved motif/domain deletion mutant strains and PH-1 on carrot medium.B,ascospores discharged from the perithecia in the bromdomain deletion mutant,complementary strain and PH-1.C,asci and ascospores produced in the bromdomain deletion mutant,complementary strain and PH-1.ΔFgGCN5,the FgGCN5 deletion mutant; ΔFgGCN5-C,the complementary strain of the gene deletion mutant; FgGCN5ΔC,the bromodomain deletion mutant; FgGCN5ΔN,the catalytic domain deletion mutant; FgGCN5E130K,E130K site mutant; FgGCN5ΔI-FgGCN5ΔIV,the motifs I-IV deletion mutants.Bar=20 μm.

Fig.8 Nuclear localization of the FgGCN5 in conidial and hyphal cells.The conidia and hyphae were stained with DNA dye Hoechst 33342,and the nuclear localization of the FgGCN5 was observed both for Hoechst 33342 and GFP.A,nuclear localization of the FgGCN5 in conidia cells.Bar=50 μm.B,nuclear localization of the FgGCN5 in hyphae cells.Bar=20 μm.

The deletion of each conserved motif in the catalytic domain of FgGCN5 resulted in multifaceted defects in the phenotypes of the mutant strains,including colony morphology,mycelium vegetative growth,conidiation,DON production,virulence and sexual reproduction.Coincidentally,the defective phenotypes of each deletion mutant were similar to those observed for full-length gene deletions (ΔFgGCN5).These results indicate that the introduction of truncated genes lacking the conserved motifs fails to recover defects of the gene deletion mutants.This suggests that each of the conserved motifs in the catalytic domain is critical to the functionality of the gene in the fungus.

GCN5 in yeast was originally characterized as a transcriptional activator through its enzymatic modification on histones (Georgakopoulos and Thireos 1992; Brownellet al.1996; Candauet al.1997).The enzymatic activity of GCN5 is required for gene transcriptional activation.However,only the catalytic activity of GCN5 is insufficient to activate gene transcription.For transcriptional activationin vivo,ADA2 binding to GCN5 is indispensable (Candauet al.1997).GCN5 forms multi-proteins complexes,such as in SAGA (Spt-Ada-Gcn5-Acetyltransferase) and ADA(alteration/deficiency in activation) (Grantet al.1997; Pray-Grantet al.2002; Leeet al.2011,2014).Accumulating evidence has highlighted the interaction between GCN5 and ADA2 in the complex.InPopulus trichocarpa,the two-subunit complex of GCN5 and ADA2 is required for transcriptional activation of PtrAREB1-2 to form the ternary-protein complex,which consequently activates NAC transcription (Liet al.2019).In yeast,the deletion of each motif in the catalytic domain leads to a loss of its interaction with ADA2,leading to a loss of gene transcription (Candauet al.1997).In this study,defects in the motif deletion mutants were likely to be attributed to the deletion of each motif in the catalytic domain,preventing protein folding and transcriptional activation (Candauet al.1997).

E130 is strictly conserved in GCN5 which acts as a proton acceptor.Thus E130 plays a critical role in catalyzing deprotonation of the substrate lysine (Linet al.1999).Mutation of this residue leads to a drastic reduction in the catalytic activity of GCN5 (Tanneret al.1999).This may explain why the E130K mutation in FgGCN5 shows a similar defective phenotype to that observed for the gene deletion mutant.

4.2.The bromodomain is important for DON production and virulence of F. graminearum

The bromodomain in the carboxyl-terminal also conserved in nuclear localized GCN5 homologs.In this study,the deletion of the bromodomain of FgGCN5 has no impact on hyphal vegetative growth,asexual or sexual development,or nuclear localization of the gene in conidia and hyphae cells (data not known).However,DON production and virulence were lower in the bromodomain deletion mutant compared to the wild type.These results indicate that the bromodomain mediates pathogenesis but plays no role in the development of the fungus.As a major virulence factor ofF.graminearum,DON is important for invasive hyphae spread during infection on wheat heads (Proctoret al.1995).Increasing evidence had confirmed the role of DON production on pathogen virulence.For example,the deletion ofFgPEX1,FgPEX4,andFgCrz1A,results a loss of DON production and decreased virulence (Chenet al.2019; Zhanget al.2019a,b).Trigenes play an essential role in DON biosynthesis.In this study,DON production was blocked in each motif (I-IV) deletion mutant and E130K site mutants,and the transcription ofTri5,Tri6andTri10genes in the six mutants were similar to those of the gene deletion mutant which are consistent with the results of Konget al(2018).However,in the bromodomain deletion mutant,the threeTrigenes were expressed at the same level as the wild type.These results indicate that the bromodomain in FgGCN5 makes no effect on theTrigenes transcription.Though the transcriptions of theTrigenes were regular,DON production was lower in the bromodomain deletion mutant.It is therefore reasonable to speculate that damage occurs downstream of DON biosynthesis or that DON secretion outside of the cells is limited.

5.Conclusion

In summary,our data indicate that FgGCN5 is localized to the nucleus.The four conserved motifs in the catalytic domain and the E130 residue are all essential to the correct roles ofFgGCN5.The bromodomain in the carboxyl-terminus plays an important role in DON production and virulence of the pathogen,but is dispensable for colony morphology,hyphal growth,asexual and sexual development,and the nuclear localization of the fungus.

Acknowledgements

This study was supported by the open project of the State Key Laboratory of Crop Stress Biology for Arid Areas,Northwest A&F University,China (CSBAA2016001).